Vaccine for fish cold-water disease

ABSTRACT

The present invention provides a vaccine for the bacterial cold-water disease in fish comprising inactivated cells of  Flavobacterium psychrophilium  in the logarithmic growth phase or components thereo

FIELD OF THE INVENTION

The present invention relates to a vaccine against (bacterial)cold-water disease in fishes and a method for preventing the disease infish using the vaccine.

BACKGROUND OF THE INVENTION

Cold-water disease is a disease occurring in salmon, trout, ayu(sweetfish) and crucian carp in low water temperature seasons. Thisdisease, which attacks young fish in low water temperature seasons andhas a high mortality, was originally discovered in trout in NorthAmerica. While the mortality rate is 20 to 50%, another problem is thatsequelae such as ulcers remain on the surface of the fish that haveescaped death.

Although therapy for cold-water disease include raising the watertemperature or oral administration of sodium sulfizole, raising thewater temperature above 25° C. is uneconomical treatment whileadministration of drugs is not preferable for edible fish.

It has been proved that the pathogen of the cold-water disease isFlavobacterium psychrophilium, which is also known as Flexibactorcyclophils or Cytophagar cyclophils. However, no vaccines against thisdisease have yet been developed.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide thevaccine against cold-water disease in fish.

The inventors of the present invention have investigated Flavobacteriumpsychrophilium as a pathogen of the cold-water disease in terms ofpathogenicity and vaccine activity depending on various cultivationconditions, and found a quite unexpectedly that the vaccine activitybecomes higher by using bacteria in a logarithmic growth phase ratherthan by using bacteria in a stationary-state phase. The presentinvention has been completed based on this findings.

In a first aspect, the present invention provides the vaccine forcold-water disease in fish comprising inactivated cells ofFlavobacterium psychrophilium in a logarithmic growth phase orcomponents of the cells.

In a second aspect, the present invention provides the vaccinecomposition for cold-water disease in fish containing inactivated cellsof Flavobacterium psychrophilium in a logarithmic growth phase orcomponents of the cells.

In a third aspect, the present invention provides the method forpreventing cold-water disease in fish comprising administering aneffective dosage of inactivated cells of Flavobacterium psychrophiliumin a logarithmic growth phase or components of the cells.

It may be concluded that cold-water disease of salmon, trout, carp andayu can be efficiently prevented by using the vaccine of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the relationship between the culture time andoptical density (OD) at 600 nm and the number of cells (CFU/mL);

FIG. 2 is a graph showing the pathogenicity (accumulated mortality) ofayu depending on the culture conditions of the bacteria of the presentinvention;

FIG. 3 shows the results of the SDS-PAGE analysis of the cell componentsof the bacteria of the present invention;

FIG. 4 shows scanning electron microscope photographs (A, C andE=×20,000 magnification; B, D and F=×100,000 magnification) of thebacteria of the present invention at logarithmic growth phases (A and B:36 hours) and at stationary phases (C and D: 48 hours, E and F: 72hours);

FIG. 5 shows transmission electron microscope photographs of ultra-thinslices of the bacteria of the present invention in the logarithmicgrowth phase.

FIG. 6 shows scanning electron microscope photographs of the lower jawof ayu infected with the bacteria of the present invention;

FIG. 7 shows the survival rate in challenge 1 (challenge 3 weeks afteradministration of the vaccine);

FIG. 8 shows the survival rate in challenge 2 (challenge 7 weeks afteradministration of the vaccine);

FIG. 9 is a photograph showing the symptoms of the dead ayu (the arrowsshow the symptoms specific to the cold-water disease); and

FIG. 10 is a photograph showing the results of diagnosis of infection,if any, of dead ayu with the bacteria of the present invention detectedby a fluorescent antibody test;

FIG. 11 is a graph showing the pathogenicity (accumulated mortality) ofthe bacteria of the present invention against rainbow trout; and

FIG. 12 shows photographs of healthy rainbow trout in the control group(A), symptoms of dead rainbow trout that died on day 1 after challengeby immersion (B, C and D), symptoms of dead rainbow trout that died onday 5 after challenge by immersion (E and F), and Flavobacteriumpsychrophilium found in the caudal fins of dead rainbow trout.

BEST MODE FOR CARRYING OUT THE INVENTION

Inactivated cells of Flavobacterium psychrophilium (may be referred toas the bacteria of the present invention hereinafter) in a logarithmicgrowth phase or components of the cells are used in the vaccine of thepresent invention. Usually, bacterial cultivation phases can be dividedinto a lag phase, logarithmic growth phase, stationary phase, extinctionphase and survival phase. Many projections were observed on the surfaceof invading bacterial cells upon observation of the bacterial cells ofthe present invention invading fish bodies. On the other hand,differences of cell secretory products were detected by SDS-PAGE and theexistence of projections was observed on the surface of the bacterialcells in the logarithmic growth phase upon observation of theconfiguration and analysis of the bacteria of the present invention inthe lag phase, logarithmic growth phase and stationary phase.

The bacterial cells of the present invention used for production of thevaccine are obtained by cultivating the cells according to conventionalmethods and by harvesting the cells in the logarithmic growth phase. Thebacterial cells of the present invention may be inoculated on anappropriate culture medium and cultivated according to conventionalmethods. The culture medium preferably contains an appropriate amount ofassimilable carbon and nitrogen sources.

The carbon and nitrogen sources are not particularly restricted.Examples of them include tripton, serum of various animals, corn glutenmeal, soy bean powder, corn steep liquor, casamino acid, yeast extract,pharma media, sardine meal, meat extract, peptone, HiPro®, AjiPower®,corn meal, soy bean meal, coffee refuse, cotton seed oil refuse,Cultivator®, Amiflex® and Ajipron®, Zest® and Ajix®. Examples of thecarbon source include assimilable carbon sources such as arabinose,xylose, glucose, mannose, sucrose, maltose, soluble starch, lactose andcane molasses, and assimilable organic acids such as acetic acid.Phosphates, organic salts such as Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺, Co²⁺, Na⁺ andK⁺ salts, and other inorganic salts and trace amounts of nutrients, ifnecessary, may also be added to the culture medium. Commerciallyavailable culture media such as TY culture medium and Cytophagar (CYT)culture medium, as well as modified Cytophaga (MCYT) culture medium andculture medium supplemented with bovine fetal serum may also be used.

The culture condition is preferably controlled at pH 6.8 to 8.4 and at atemperature of 4 to 20° C.

Whether the bacteria of the present invention are in the logarithmicgrowth phase or not may be confirmed by measuring the optical density at600 nm, which dramatically increases in the logarithmic growth phase.For example, cultivation reaches the logarithmic growth phase after 20to 30 hours' cultivation at pH 7.3 and 15° C.

The bacteria of the present invention in the logarithmic growth phaseare separated by centrifugation or filtration, or the culture productmay be directly inactivated. The inactivation treatment includes heattreatment or formalin treatment.

The bacteria of the present invention contain cell membrane components,vesicles and secretary products. These components are preferablycollected after ultrasonic pulverization of the inactivated bacterialcells.

The inactivated bacterial cells and components thereof are preferablyused after filtration, or after concentration by evaporation orlyophilization.

Although the inactivated bacterial cells of the present invention may bedirectly used as the vaccine, they may be formulated into a vaccinecomposition together with a pharmaceutically acceptable liquid or solidcarrier. Examples of the formulation of the vaccine composition includeoral administration compositions, injection compositions, compositionsfor immersing fish, and feed compositions. Examples of the liquidcarrier include water and physiological saline, while examples of thesolid carrier include excipients such as talc and sucrose. Theinactivated bacterial cells of the present invention or componentsthereof may be mixed with conventional fish feeds to prepare the feedcomposition. An adjuvant may be added to these vaccine compositions inorder to enhance the antigenicity.

While the vaccine or vaccine composition of the present invention may beadministered to adult fish, it is preferably administered before theonset of cold-water disease, for example, during the period when thefish is young. The dosage is preferably about 1 mg to 5 g per 1 kg ofthe body weight as converted into the weight of the inactivatedbacterial cells or components thereof. The dosage may be once or severaltimes, for example 2 to 10 times. The vaccine may be administered everyday, or with an interval of 1 to 2 days.

The fish that can be administered the vaccine or vaccine composition ofthe present invention are not particularly restricted so long as thefish are afflicted by cold-water disease caused by the bacteria of thepresent invention; examples of the fishes include ayu (sweetfish) andcrucian carp, and salmon and trout such as yamame (salmo masau), rainbowtrout and silver trout.

EXAMPLES

While the present invention is described in more detail hereinafter withreference to examples, the present invention is by no means restrictedto these examples.

Example 1

(1) Cells of Flavobacterium psychrophilium G3724 (this strain was usedin the experiments hereinafter) contained in a platinum loop wereinoculated on a 4-mL MCYT culture medium (trypton 2.0 g, yeast extract0.5 g, meat extract 0.2 g, sodium acetate 0.2 g, calcium chloride 0.2 g,distilled water 1000 mL, pH 7.2). After cultivation at 15° C. for 2days, a 0.5-mL fraction of the culture medium was inoculated on a 200-mLMCYT culture medium followed by cultivation with shaking at 15° C. Therelationship between the cultivation time, and the cell number andoptical density at 600 nm is shown in FIG. 1. FIG. 1 shows that the lagphase is from 0 to 24 hours after inoculating, the logarithmic growthphase is 24 to 48 hours after inoculating, and the stationary phase isafter 48 hours from inoculating in the bacteria of the presentinvention.

(2) The differences in pathogenicity of the bacteria of the presentinvention depending on the culture conditions were investigated. Thebacteria of the present invention in the logarithmic growth phase andstationary phase were added to an aquarium of ayu at a concentration of10⁸ to 10¹⁰ CFU/mL to determine the pathogenicity of the bacteria. Ayuused for the experiment had a body weight of 0.5 to 5 g, and thetemperature of the aquarium was 15° C. As shown in FIG. 2, while themortality rate of the fish in the infection group using the bacteria ofthe present invention in the stationary phase until day 10 of theexperiment was 20 to 60% of the mortality rate of the fish in thecontrol group (non-infection group), the mortality of the fish in theinfection group using the bacteria of the present invention in thelogarithmic growth phase at day 10 of the experiment was 100%, showingthat the bacteria in the logarithmic growth phase have higherpathogenicity than the bacteria in the stationary phase.

(3) The bacterial cells of the present invention in different growthphases were pulverized by ultrasonic waves. Each fraction of the extractwas isolated by sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE, silver staining). The results are shown in FIG. 3. Theresults show that certain substances are produced specifically in thelogarithmic growth phase (indicated by arrows in the graph).

(4) The bacteria of the present invention in the logarithmic growthphase and stationary phase were observed under a scanning electronmicroscope (FIG. 4) and transmission electron microscope (FIG. 5). Itwas revealed from the results that projections can be seen on thesurface of the bacterial cells in the logarithmic growth phase.

(5) Ayu were infected with the bacteria of the present invention in thelogarithmic growth phase. It was observed under the scanning electronmicroscope that the bacteria of the present invention had invaded intothe lower jaw of the ayu (FIG. 6). The result indicates that thebacteria of the present invention in the logarithmic growth phase havingthe vesicles invaded into the body of the ayu.

Example 2

Flavobacterium psychrophilium G3724 was cultured in 1000 mL of the MCYTculture medium contained in a 2000-mL Sakaguchi flask at 15° C. Thecells showing OD 0.2 to 0.7 at 600 nm were used as the bacterial cellsin the logarithmic growth phase. Then, the cells as a culture product ata growth phase showing OD of 0.2 to 0.7 at 600 nm in the culture periodof 24 to 36-hour were inactivated by incubation in 0.3% formalin at 15°C. for 2 days, and the inactivated bacterial cells were isolated bycentrifugation at 4° C. and 8,000 to 10,000×g. The bacterial cells inthe stationary phase after 36-hour cultivation (OD_(600 nm)=1.0) werealso inactivated by the same method as described above to obtaininactivated bacterial cells as controls.

Example 3

Cells of Flavobacterium psychrophilium G3724 contained in a platinumloop was inoculated on 50 mL of the MCYT culture medium and pre-culturedat 15° C. for 48 hours. A 2.5-mL fraction of this culture medium wasinoculated on 1000 mL of the MCYT culture medium, followed by culture at15° C. for 36 hours. OD at 600 nm was 02 to 0.7. The culture product wasincubated in 0.3% formalin at 15° C. for 2 days. The bacterial cellswere then collected by centrifugation at 8,000 to 10,000×g at 4° C. Thecells obtained were re-suspended in physiological saline containing 0.3%formalin to obtain a vaccine suspension containing the inactivatedbacterial cells of the present invention.

Example 4

The inactivated bacterial cells obtained from the cells in thelogarithmic growth phase and stationary phase in Example 2 were orallyadministered to ayu with an average body weight of 5.0 g at a dosage of0.1 FKCg/kg/day.

After the oral administration as described above, the ayu werechallenged by immersing in the bacterial solution. The results are shownin Table 1. TABLE 1 Dosage of Challenge Death/ Survival Group (CFU/mL)Challenge Rate (%) Logarithmic Growth Phase Group 1.7 × 10⁸ 39/15274^(a,c) Stationary Phase Group 1.9 × 10⁸ 39/105 63^(b) Control Group2.2 × 10⁸ 82/165 50^(a)Significant difference against control group (p < 0.001), chi-squaretest^(b)Significant difference against control group (p < 0.05)^(c)Significant difference against stationary phase group (p < 0.05)

Table 1 shows that the difference in the survival rate was significantin both the stationary phase group and logarithmic growth phase group ascompared with the control group. However, the survival rate of thelogarithmic growth phase group was significantly higher than that of thestationary phase group, showing that the logarithmic growth phase groupis particularly useful as the vaccine.

Example 5

The effect of the vaccine was investigated using the vaccine compositionobtained in Example 3. The vaccine was orally administered for 2 weeks(0.1 g/kg) to the fish from 5 weeks before the start of challenge, andthe fish were fed on a standard feed for 3 weeks to enhanceimmunological activity. The fishes were then divided into two groups:one in which the challenge was started 3 weeks after the end of vaccineadministration, and another in which the challenge was started 7 weeksafter the end of vaccine administration.

Two thousand “ayus” with a body weight of 0.5 g were divided into twogroups. The vaccine was either orally administered every day to thefishes in one group, or five times in two weeks (oral administrationwith an interval of two days) to the fishes in the other group. Theresults are shown in Table 2, and in FIGS. 7 and 8. TABLE 2 AverageAmount of No. of Body Challenge Deaths/No. of Survival Weight (g)(CFU/mL) Challenges Rate (%) Challenge 1^(a) 1 1.7  7/118 94.1^(b) 2 1.84.4 × 10⁷  4/119 96.6^(b) Control 1.8 36/117 69.2 1 1.9 53/114 53.5^(b)2 1.8 1.2 × 10⁸ 10/120 91.7^(b) Control 1.9 79/121 34.7 Challenge 2^(a)1 2.7 26/186 86.6^(b) 2 2.9 2.1 × 10⁷ 20/168 88.1^(b) Control 2.7 41/17476.4 1 2.7 40/170 76.5^(b) 2 3.0 1.4 × 10⁸ 36/165 78.8^(b) Control 3.2107/185  42.2^(a)Challenge 1: challenged 3 weeks after administration of vaccine,Challenge 2: challenged 7 weeks after administration of vaccine^(b)significant difference against control group (p < 0.01)1: the group in which the vaccine was administered every day for 2 weeks2: the group in which the vaccine was administered 5 times in 2 weeks

The results of the challenge tests three weeks after the administrationof the vaccine show that a significant difference was observed betweenthe vaccine-administered group and control group. It was also shown thatthe effect of the vaccine is higher in the group in which the vaccinewas administered only five times than in the group in which the vaccinewas administered every day.

The effect of the vaccine was significantly higher in bothvaccine-administered groups than in the control group, when thechallenge test was performed 7 weeks after administration of thevaccine.

In the test fish that died in the test period of the present invention,it was confirmed whether the death was ascribed to the bacteria of thepresent invention or not. As shown in FIGS. 9 and 10, typical symptomsof the cold-water disease were observed in all the dead fish. It wasalso revealed that the cause of death of the test fish during the testperiod of the present invention was infection with the bacteria of thepresent invention, since staining of the dead fish with a fluorescentantibody was positive with respect to all the individuals tested.

Example 6

Flavobacterium psychrophilium NCMB 1947 was cultured with shaking inMCYT culture medium at 15° C., and the culture medium in the logarithmicgrowth phase was used for artificial infection when OD600 during 24 to48 hours' cultivation reached 0.2 to 0.7. The bacteria of the presentinvention in the logarithmic growth phase were added to an aquarium ofrainbow trout so that the concentration of the bacteria was 10⁶ to 10⁸CFU/ml to attempt artificial infection by the immersion method. The bodyweight of rainbow trout used for the experiment was in the range of 1 to4 g, and the water temperature was 15° C. As shown in FIG. 11, themortality rate of the fish in the group infected with the bacteria ofthe invention in the logarithmic growth phase was 55.8%, in contrast to0% in the control group (non-infection group). This result is the firstsuccessful artificial infection of rainbow trout by the immersionmethod. The photographs in FIG. 12 show healthy rainbow trout, symptomsof rainbow trout that died on day 1 (B, C and D) and on day 5 (E and F),and Flavobacterium psychrophilium found in the caudal fins of deadrainbow trout (G and H).

1. A vaccine against the cold-water disease in fish, comprising, as aneffective component, inactivated cells of Flavobacterium psychrophiliumin a logarithmic growth phase or components of the cells.
 2. A vaccinecomposition for the cold-water disease in fish, comprising inactivatedcells of Flavobacterium psychrophilium in a logarithmic growth phase orcomponents of the cells.
 3. A method for preventing the cold-waterdisease in fish, comprising administering an effective dosage ofinactivated cells of Flavobacterium psychrophilium in a logarithmicgrowth phase or components of the cells.